MCB104 - Lecture 12 - Gene Regulation

NO EPIGENETICS

What is the difference in prokaryotic vs. eukaryotic gene regulation?

What are enhancers and why do they matter?

• Unlike prokaryotes, eukaryotes have chromatin

  • Chromatin must be cleared from promoters for transcription

• Done by various chromatin remodeling proteins

• These often bind to enhancers

  • DNA sequences away from the promoter that stimulate transcription

  • Can be very distant, range is 100bp-1Mbp, but typically 1-100 kbp

  • Can be up or downstream of the promoter

What is Translation Initiation?

• Bacterial mRNAs are often multicistronic

  • Have multiple ribosome binding sites (RBSs) which means multiple polypeptides

  • Small ribosomes subunit initiates by binding the RBS

  • In Eukaryotes (typically) only one start site

  • Small ribosome subunit binds 5’ cap first and scans to the start

What is mRNA processing?

• Eukaryotes require splicing of primary transcript due to pressure of introns

  • Prokaryotes typically no introns

• Eukaryotes, but not prokaryotes, add methylated cap (5’ end) and poly-A tail (3’ end)

What is alternative splicing?

• Alternative splicing produces different mRNAs from the same primary transcript

• EXON 6…

• If the sequence required to stick in membrane (start/stop transfer sequence)

• If in Exon 7 or 8

• For VerA: 6a gets cut to 7 (membrane bound anitbody)

• For VerB: If regulatory protein it blocks splicing to occur and the A is the PolyA sequence and instead, 7 & 8 gone

• Now you have all the stuff that would have been here is in the antibody but it’s no longer attached to membrane and can be secreted

What is the 5’ methylated cap?

• Capping enzyme adds a methylated “backward” G to the 1st nucleotide of a primary transcript

What is the transcription product?

• Primary transcript is the single strand RNA result of transcription

  • In prokaryotes, primary transcript is mRNA

  • In eukaryotes, primary transcript is processed to make an mRNA

    • 5’ methylated cap

    • 3’ poly-A tail, 100-200 A bases

    • Introns removed by RNA splicing

Where does regulation of gene expression occur?

EVERY

• Regulation can occur at every step of the expression process

  • Chromatin structure

  • Transcription initation

    • Recruitment/blocking of RNA polymerase

  • Co and post transcriptional: mRNA processing

    • Splicing, base modifications, etc

  • Pre-translational: mRNA stability and avaliability

  • Translation initation

  • Post translational modifications

    • Phosphorylation, glycosylation, etc

What is regulatory elements?

DNA sequences that are NOT transcribed but play a role in regulating other nucleotides sequences

• Promoters, enhancers, silencers, etc

• CIs regulatory element: a regulatory element on the same chromosome as the target

What are regulatory genes?

Genes whose products interact with other sequences and alter their transcription or translation

• Trans acting proteins: A regulatory protein whose target can be on a different chromosome

What is Constitutively expression?

Continuously expressed under normal conditions

What is positive control?

Stimulate gene expression

What is negative control?

Inhibit gene expression

What are DNA binding proteins?

~60-90 amino acids, responsible for binding to DNA, typically via hydrogen bonds

• Motif: a simple structure that occurs frequently

• DNA binding motifs typically interact with the major groove

• Distinctive types of DNA-binding proteins based on the motif

3 examples:

What are operons?

• Bacterial gene expression often uses operons

  • Opersons consist of a promotor, regulatory sequences (operator) and structural genes

• Often controlled by regulatory genes that are not part of the operon

What is inducible operons?

Transcription is usually off and needs to be turned on

• Negative inducible operons have an inducer inactivate a repressor

• Typically catabolic pathways

What are repressible operons:

• Transcription is normally on and needs to be turned off

  • Typically anabolic pathways

What is the E.coli Lactose Metabolism

• Permease symporter transports lactose into the cell

• Lactose is hydrolyzed by beta-galactosidase

  • Often called just B-gal

• Products are glucose and galactose

How is lactose used as a gene regulation model?

• The lac genes are not essential for survival

  • If both glucose and lactose are present, E.coli cells will use glucose first

• Simple assays for lac expression ONPG or X-gal as substrates for B-gal produce colored products

• Lactose induces a 1000-fold increase in B-gal activity

• Many lac- mutants have been identified

DESCRIBE THE LAC OPERSON COMPONENTS AND PROTEINS

• Lac Operon has several components:

  • 3 Structural Genes:

    • LacZ: B-galactosidase

    • LacY: Permease

    • LacA: Acetyltransferase

  • Promotor (lacP): RNA Polymerase binding site

  • Operator (lacO): Binding site for lac repressor

  • CRP binding site: BInding site for CRP

• There are 2 regulatory proteins:

  • lacl: encodes lac repressor protein

  • CRP: cAMP receptor protein

    • Also called CAP: Catabolite Activated Protein

How does Lac operon repression work?

• The lac operon is normally inactive

• The lac repressor protein binds to LacO

• The Operator is located overlaps with the RNA polymerase binding site and covers the transcription start site

How does the lac repressor work?

• lac repressor is a tetramer, with each subunit containing a DNA-binding HTH motif

• lac operson has 3 operators (O1, O2, O3) each of which contains 2 recognition sequences for lac repressor

  • O1 has the strongest binding affinity for lac repressor

  • Maximum repression occurs when all 4 repressor subunits are bound (cooperatively)

  • 2 repressor subunits bind to O1

  • 2 repressor subunits bind to O2 or O3

How does induction of the lac operon work?

• When inducer (lactose or IPTG) is present

  • Inducer binds the lac repressor

  • Allosteric inhibition of the repressor

    • Reversibly changes shape and cannot bind to the operator

  • RNA polymerase may now bind the promoer

How does glucose and the lac operon relate?

• When both glucose and lactose are present, only glucose is used

• When glucose is low, adenyl cyclase has higher activity

• cAMP binds to CRP proteins

• cAMP-CRP binds to CRP binding sites and recruits RNA polymerase

  • Without CRP bound, recruitment of RNA polymerase is very inefficient

• Catabolite repression: the product of the catabolic pathway blocks the expression of genes involved

Describe lac mutant analysis

• Mutants reveal roles of lac operon components

• Table shows B-gal activity under different conditions

• Partial diploidy via introduction of a plasmid can show cis vs. trans activity

  • For a table, the plasmid contains all components except lacZ

  • (lacZ minus plasmid that has lacI-, if I mutate the operator (LacO-) i will have constitutent actvity but if LacI- can operate in trans and can inhibit operon even if not on same DNA sequence)

• LaclS is a gain of function that cannot be bound by inducer

What is the Trp Operon?

• Anabolic synthesis of tryptophan (Trp) is regulated by the trp operson

• When tryptophan is present, it binds to the repressor

• Trp bound repressor can bind to TrpO, blocking transcription

What is the Trp Attenuator

• The trp RNA leader can fold into 2 different conformations

  • The 3 to 4 stem loop is a transcription terminator

  • The 2 to 3 stem-loop prevents formation of the 3 to 4 stem - loop so it is an antiterminator

• A terminator may halt transcription or block the ribosome

How is the attenuator regulated by avaliability of tRNATrp

• RNA leader includes 2 trp codons

• When tRNATrp is present rapid ribosome movement results in 3 to 4 stem loop formation, which is a terminator

• When tRNATrp is low, the ribosome stalls, allowing the 2-3 loop to form, blocking the 3 to 4 loop

How does riboswitch work?

• RNA leader with several conformations

  • Binds small molecule through a short sequence called an aptamer

  • Conformation of the “expression platform” changes if aptamer is bound or unbound

• A riboswtich can regulate transcription or translation

How is gene regulated by small RNAs? (sRNA)

• Usually inhibit translation by base-pairing with RBS

• Can also activate translation by disrupting stem loops

• Some lead to mRNA degradation

How is gene regulated by antisense RNA?

• Produced by transcription of the strand of DNA opposite the template strand (antisense)

  • May inhibit translation by base pairing with the same strand

  • May lead to degradation of mRNA

  • May interfere with transcription of sense gene

What is Eukaryotic Gene Expression?

• Many steps can be regulated to control the amount of active gene product

  • Transcription initiation

  • Transcript processing

  • Export from nucleus

  • Translation of mRNA

  • Protein localization

  • Protein modification

What is Eukaryotic Gene Regulation: Cis elements - PROMOTERS

• Promotors: DNA sequence that is usually directly adjacent to the gene

  • Bind RNA polymerase

  • Often have TATA box (TATA (T/A) A(A/T)

  • Allow basal level of transcription

What is Eukaryotic Gene Regulation: Cis elements - ENHANCERS

• DNA sequence that can be far away from gene

  • Augment or repress the basal level of transcription

  • May be located either 5’ or 3’ to the transcription start site

  • Still function when moved to different positions relative to promoter

What are the cis elements of eukaryotic gene expression?

Enhancers and Promoters

What are reporter genes?

• Enhancers can be identified by making a conduct with

  • Putative enhancer

  • Minimal promoter

  • Reporter protein (GFP)

• An organism with that sequence will express GFP when that enhancer is active

What are eukaryotic trans factors:

Transcription Factors

What is the eukaryotic trans factors - Transcription Factors

• General term for any DNA binding protein that regulates transcription

• Binds to promoters and enhancers

• Recruit other proteins to influence transcription

• 3 types: Basal Factors, Activators, and Repressors

What are Basal Factors?

The core transcription machinery

Ordered pathway of assembly at promoter:

1) TATA-binding protein (TBP) binds to TATA box

2) TBP associated factors (TAFs) bind to TBP

3) RNA pol II binds to TAFs

What is the mediator complex?

• Transcription of many eukaryotic genes requires the mediator complex

• Mediator is a complex of more than 20 proteins

• Doesn’t bind DNA directly, bridges between

  • The promoter

  • Activator or repressor proteins at

Describe Activators

• Activators positively regulate transcription

• 2 main mechanisms:

  • Recruiting basal factors and RNA pol II to promoters

    • Often involves mediator for this

  • Recruiting coactivators to open chromatin structure

What is the activator structure?

• Activator proteins have at least 2 functional domains

• DNA binding domain: binds to specific enhancer

• Activation domain: binds to other proteins (basal factors or coactivators)

• Dimerization domain: some activators also have a domain that allow them to interact with other proteins

  • Homodimers pair with themselves

  • Heterodimers pair with another activators

  • Provides coincidence detection

What are Repressor Proteins?

• The opposite of activators but same principles

• 2 main mechanisms:

  • Recruit co-repressors to prevent RNA pol II complex binding

  • Recruit co-repressors to close chromatin structure

How can the same transcription factor play different roles?

• Transcription factors’ impacts on transcription are context dependent

• The presence or absence of other transcription factors can cause them to act as either repressors or activators

  • Ex: Dorsal is an activator unless it is near dead ringer (dri) at which point they bind the corepressor Groucho

How does transcription factor regulation work?

• The function of trans acting proteins changes by

  • Allosteric interactions (ex: Ligand binding)

  • Modificaton of transcription factors (ex: phosphorylation)

  • Transcription factor cascades

How does fine-tuning gene expression work?

• In humans, approximately 2000 genes encode transcriptional regulatory proteins

• Each regulatory proteins can act on many genes

• Each enhancer has binding sites with varying affinities for activators and repressors

• Post-translational modifications and ligand binding can alter TF activity

What are indirect repressors?

An indirect repressor interferes with the function of an activator

• Competition due to overlapping binding sites

• Repressor binds to activation domain (quenching)

• BInding to activator and keeping it in cytoplasm

• Binding to activator and preventing homodimerization

What are insulators?

• Insulators are sequences located between an enhancer and a promoter that block access to the promoter

• Human insulators bind CTCF proteins to form loops called topologically associating domains (TADs)

• Enhancers activate promoters located in the same loop

How do you detect TADs?

• Chromatin Conformation Capture:

• Cross-link proteins and DNA in close proximity within chromatin

• DNA is fragmented and DNA ligase joins ends

• High-throughput methods used to define TADs

  • Measures frequency with which 2 sequences were ligated together

How can you study transcription factors using reporters?

• GFP reporters can be used to screen for mutations in transcription factors

• Mutations in a gene encoding in an activator reducing expression of the reporter

• Mutations in a gene encoding a repressor increase expression of the reporter

How can you study transcription factors using CHIP-Seq?

• Co-immunoprecipitation: Use an antibody against a target (Bait) protein to isolate it and anything attached to it (Prey) for analysis

• Chromatin immuniprecitation: Co-IP where the bait is a TF and the prey is the DNA

• Steps:

  • Crosslink DNA

  • Fragment DNA

  • Bind with an antibody specific to the target protein

  • Purify complexes with antibody, target protein, and DNA fragments

  • Sequence DNA

How does posttranscriptional regulation work?

• Posttranscriptional regulation can occur at many steps

• At level of RNA:

  • Splicing, stability, and localization

• At level of protein:

  • Synthesis, stabillity, modification, and localization

What is Splicing Regulation?

• Fruitless (fru) in Drosophila is required for courtship

• Both male and female flies produce same primary transcript

• Females express Transformer (Tra) protein that remove an exon from the final transcript

• Result is sex specific Fru proteins

How can translation initiation be regulated?

• Control of translation often occurs at initiation

  • Small subunit of ribosome recognizes a complex structure around 5’ cap

  • eIFG protein binds to poly-A binding protein (PABP) at poly-A tail to circularize mRNA

• Example Regulation:

  • 4E-BP1 binds to initiation elF4E, blocks initiation

  • Presence of nutrients and growth factors in environment leads to phosphorylation of 4E-BP1, preventing association

How does Poly-A tail length work?

• Longer poly-A tails bind PABP more efficiently

• Translation initation complex forms more efficiently

• Once tail is removed, mRNA is targeted for degradation

How does Translational Control by ORF work?

• Upstream open reading frame can be translated instead of the main ORF

• Regulators can bind the uORF to encourage or discourage its translation

How does Ribosomal Profiling work?

• mRNA level doesn’t always correlate with protein level

• Ribosome profiling allows researchers to observe positions of ribosomes on mRNAS

• Steps:

  • Purify (often crosslink) ribosome/mRNA complexes

  • Digest with RNAase to eliminate uncovered RNA

  • Sequence RNAs to determine what is being transcribed

Think of miRNAs, siRNAs, piRNAS and their targets & effects

What are miRNAs?

• Most miRNAs are transcribed by RNA polymerase II

• The primary transcripts have double stranded stem loops

• Drosha excises stem-loop from primary miRNA (pri-miRNA) to generate pre-miRNA

• Dicer processes pre-miRNA to a 21-25nt long duplex miRNA

• One strand is incorportated into miRNA-induced silencing complex (miRISC)

What is miRNA regulation?

• miRNAs bind to complementary sequences of other RNAs

• When complementarity is perfect, target mRNA is degraded

• When complementarity is imperfect, translation of mRNA target is repressed

What are siRNAs?

siRNAs follow the same pathway as miRNAs except:

• Source is different: exogenous dsRNAs or transcription of both strands of an endogenous genomic sequence

• A few different components in RISC complex

• Almost always a perfect match, so almost always degrades target mRNA

• Some riRNAs bind to genomic sequence and recruit factors that convert it to heterochomatin

The siRNA response is called RNA interference (RNAi)

RNAi is a common genetic tool, permitting the knockdown of target genes without having to make mutants

• Discovered in C. elegans

What is Drosophila Sex Determination?

• Sex lethal (Sxl) gene encodes an RNA binding protein that controls the alternative splicing of RNA targets

• Required for female-specific development

• In early embryos, Sxl is transcribed only in females

  • XX cells express Sxl from establishment promoter (Pe)

  • Genes encoding activators for Pe are on the X chromosome

  • Dose dependent: 2 copies required to meet threshold

How does Sxl regulate alternative splicing?

• After early embryogenesis, transcription of Sxl occurs from the Pm promoter

• Transcripts in males have a stop codon in exon 3

• In females, Sxl includes the exon 3, leading to protein production

What does Sxl trigger?

• Splicing “cascade”

• Sxl regulates splicing of transformer (tra) mRNA

• Tra protein regulates splicing of dsx mRNA (and many others)